Constructs and methods for the production and secretion of polypeptides

ABSTRACT

Described herein are molecules, constructs and methods for the production and secretion of polypeptides of interest by host cells, preferably bacterial host cells, and more particularly gram positive bacteria. In particular, the present invention is related to a polynucleic acid encoding a fusion protein and to uses thereof for the secretion of heterologous or homologous polypeptides of interest by a bacterial host cell, preferably  Clostridium  bacteria. The present invention further relates to methods and constructs for the production and secretion of heterologous or homologous polypeptides of interest proteins by host cells using such polynucleic acids and fusion proteins.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.13/056,352 (now U.S. Pat. No. 8,735,133), filed Jan. 28, 2011, which isthe U.S. National Phase of International Application No.PCT/EP2009/059875, filed Jul. 30, 2009, which claims priority to EPApplication No. 08291120.7, filed Nov. 28, 2008, and EP Application No.08290739.5, filed Jul. 31, 2008, each of which are incorporated byreference herein in their entireties.

FIELD OF THE INVENTION

Described herein are molecules, constructs and methods for theproduction and secretion of polypeptides of interest by bacterial hostcells. In particular, the present invention is related to polynucleicacids encoding a fusion protein and to uses thereof for the secretion ofa heterologous or homologous polypeptide of interest by a bacterial hostcell. The invention further relates to fusion proteins or parts thereofencoded by such polynucleic acid, and vectors and host cells containingsaid polynucleic acid. The present invention further relates to methodsfor the production and secretion of heterologous or homologouspolypeptides of interest proteins by bacterial host cells using suchpolynucleic acids and fusion proteins.

BACKGROUND

Secretion of heterologous proteins is a widely used technique inindustry. A cell can be transformed with a nucleic acid encoding aheterologous protein of interest to be secreted and thereby producelarge quantities of desired proteins. This technique can be used toproduce a vast amount of protein over what would be produced naturally.Proteins of interest are proteins with a wide variety of industrialapplications, including therapeutic and agricultural uses, as well asuse in foods, cosmetics, cleaning compositions, animal feed, etc. Thus,increasing secretion of proteins produced by micro-organism is ofgeneral interest.

Advances in cellular and molecular biology have made it possible, incertain cases, to identify a gene encoding a desired protein, to isolatethe gene, to insert the gene into a host cell and to express theinserted gene in the host cell to produce the desired protein. Bacteriahave been intensively studied as host cells. When bacteria are used ashost cells for this heterologous gene expression, a frequentlyencountered problem is however that most bacterial expression systemsproduce proteins intracellularly, and it is usually necessary to disruptthe cells to ensure recovery of the products.

The problem may be overcome by having the bacteria secrete the desiredprotein into the growth medium. One particularly well documented methodof directing the secretion of proteins is the use of a secretory signalsequence. When a signal peptide is fused to the amino-terminal end of aheterologous protein, it directs the heterologous protein to thesecretory machinery at the cell membrane. The heterologous protein isthen translocated across the membrane. Optionally a specific protease,sometimes referred to as “signal peptidase” or “leader peptidase”,removes the signal peptide and releases the heterologous protein.

Translocation of proteins into periplasmic space or secretion into theirculture media is subject to a variety of parameters. Typically, vectorsfor secretion of a protein of interest are engineered to position DNAencoding a secretory signal sequence 5′ to the DNA encoding the proteinof interest. To increase secretion several approaches can be followed:trying several different signal sequences, mutating the signal sequence,or altering the secretory pathway within the host. However, in manycases the amount of heterologous protein secreted when making use ofonly a signal peptide to ensure secretion is usually very small, and asignificant amount of the heterologous protein is often degraded afterit is secreted.

Clostridium is a genus of Gram-positive bacteria, which is representedby a wide variety of strains. Clostridium bacteria are spore-forminganaerobic bacteria. This genus comprises solventogenic Clostridia suchas C. acetobutylicum that are able to convert various sugars andpolysaccharides into acids and solvents, and cellulolytic Clostridia,such as Clostridium cellulolyticum, that are able to efficiently degradecellulose and related plant cell wall polysaccharides. More inparticular, Clostridium cellulolyticum produces and secretes largecellulolytic complexes called cellulosomes that efficiently degradecellulose and related plant cell wall polysaccharides. These complexescontain various enzymes which are tightly bound to a large proteindevoid of enzymatic activity called “scaffoldin”. The binding of theenzymes on the scaffoldin occurs through interaction between cohesionmodules on the scaffoldin and complementary dockerin domains on theenzymes. This high affinity interaction between dockerins andscaffoldins has been suggested for biotechnology applications e.g.recombinant protein purification (Craig et al. 2005, J. Biotechnol.121:165-173).

On the contrary, C. acetobutylicum although it contains in its genomecontains a large cluster of genes encoding cellulolytic enzymes and ascaffoldin, is not able to grow on crystalline cellulose.

One of the strategies to combine cellulose-degrading activity withsolvent production in one organism has been to introduce the genesencoding the cellulosome of C. cellulolyticum into C. acetobutylicum.Mingardon et al. have demonstrated the production, assembly andsecretion of a minicellulosome by Clostridium acetobutylicum byco-expressing the Mannanase gene Man5K from Clostridium cellulolyticumwith the gene cipC1 encoding a truncated scaffoldin also from C.cellulolyticum therein (Mingardon et al. Applied Environm. Microbiol.2005, vol 71(3): 1215-1222).

Several groups have investigated the possibility of increasing orimproving the cellulolytic activity of cellulosome complexes by playingwith the different modules present therein and combining different typesof cellulases in what is referred to as “designer cellulosomes”. It wasdemonstrated that bifunctional and trifunctional designer cellulosomeswhich include a chimeric scaffoldin with two or three cohesins ofdivergent specificity and two or three cellulases each bearing adockerin complementary to one of the cohesins yielded a multiproteincomplex with enhanced synergistic activity on recalcitrant substratessuch as straw (Fierobe et al. 2002, J. Biol. Chem. 277, 49621-19630;Fierobe et al. 2005, J. Biol. Chem. 280(16):16325-16334). In addition itwas found that such cellulosomes could include combinations of bacterialand fungal enzymes (Mingardon et al. 2007, Appl. Environm. Microbiol.73(12):3822-3832). In these experiments the cellulosomes were eitherproduced by co-expression of the vectors encoding the different parts ofthe cellulosome in Clostridium cellulolyticum which naturally secretesthese proteins or by mixing the recombinantly produced and purifiedscaffoldins and enzymes in vitro.

Mingardon et al. describes the production of a “covalent cellulosome”,which comprises, in a single polypeptide chain, a CBM together with afamily 48 and a family 9 catalytic module. This protein was recoveredfrom E. coli in which it was overexpressed by breaking the cells in aFrench press and purifying the recombinant protein using the c-terminalHis tag. The covalent cellulosome was found to be significantly lessactive on Avicel substrate than the corresponding hybrid cellulosomes(Mingardon et al. 2007, Appl. Environm. Microbiol. 73(22):7138-7149).

Cloning of heterologous or homologous genes encoding secreted proteins,and (over)production and secretion of such heterologous or homologousproteins by bacterial cells such as Clostridium species other than C.cellulolyticum has not been very widely reported up until now, probablyas a result of problems encountered with ensuring secretion ofrecombinant proteins by these hosts.

In view of the above, it is clear that there is a need in the art toimprove secretion of proteins by bacterial cells.

SUMMARY OF THE INVENTION

The instant invention aims to provide an approach to produce and secreteheterologous polypeptides of interest by a bacterial cell, moreparticularly a gram-positive bacterial cell and/or to improve theproduction and secretion of homologous polypeptides of interest by agram positive bacterial cell, and in particular in a Clostridiumbacterium. Also provided herein are novel molecules and constructsuseful in the methods of protein secretion provided herein, and methodsof making such molecules and constructs.

The present application is at least partly based on the finding of a newmethod for microbial production and export of a polypeptide of interestwhich avoids at least some of the problems associated with secretion asenumerated above. The molecules, constructs and methods according tothis invention make it possible to (over)produce and secretepolypeptides of interest, by a bacterial cell. In particular, thepresent invention provides a polynucleic acid encoding a fusion protein,wherein said fusion protein has a carrier domain which has a functionaleffect on the secretion of the fused polypeptide of interest. More inparticular, the inventors have shown a functional effect of a carrierdomain of a fusion protein, i.e. the capability of controlling (inducingand/or improving) (extracellular) secretion of a homologous or aheterologous polypeptide of interest by a recombinant host cell,producing said fusion protein. Said carrier domain comprises acarbohydrate binding module (CBM) and a hydrophilic module (X module),typically of a scaffolding protein and, more particularly in combinationwith a secretion signal peptide ensures (improved) secretion of apolypeptide of interest. As such the present invention thusadvantageously also provides for the use of at least a part of ascaffolding protein, and in particular at least the modules including aCBM, a hydrophilic module thereof, in particular in combination with asignal peptide, for controlling secretion in a host cell of a homologousor a heterologous polypeptide of interest fused to said part of thescaffolding protein.

In a first aspect, the invention therefore provides a polynucleic acidencoding a fusion protein consisting of a polypeptide sequence whichcomprises in this particular order:

-   -   a carrier domain comprising at least one carbohydrate binding        module (CBM) of a cellulosomal scaffolding protein fused to at        least one hydrophilic domain of a cellulosomal scaffolding        protein;    -   at least one peptide linker for linking the carrier domain to        the polypeptide of interest, and    -   at least one polypeptide of interest.

In a particular embodiment of the invention said polynucleic acidfurther comprises an in frame nucleic acid sequence for the secretion ofthe encoded fusion protein, and preferably said nucleic acid sequenceencodes a signal peptide of a cellulosomal scaffolding protein.

Accordingly, the invention provides polynucleic acids encoding a fusionprotein consisting of a polypeptide sequence which comprises, and moreparticularly, in this order:

-   -   at least one suitable signal peptide    -   a carrier domain comprising at least one carbohydrate binding        module (CBM) of a cellulosomal scaffolding protein fused to at        least one X module of a cellulosomal scaffolding protein;    -   at least one peptide linker for linking the carrier domain to a        polypeptide of interest; and    -   at least one polypeptide of interest.

In particular embodiments of the invention the peptide linker comprisesa protease cleavage site for the cleavage of said polypeptide ofinterest from the remaining fusion protein.

In further particular embodiments, the polypeptide sequence comprisestwo or more X modules, more particularly two X modules.

In another aspect, the invention is directed to the use of a carrierdomain as defined herein, more particularly in combination with a signalpeptide, for controlling the secretion of a polypeptide of interest,preferably a polypeptide as defined herein, by a host cell.

In another aspect, the present invention relates to a vector comprisinga polynucleic acid according to the invention. Preferably a vector isprovided wherein the polynucleic acid is under the control of regulatorysequences for expression of the nucleic acid in a bacterial cell.

In yet another aspect, the invention provides a host cell comprising apolynucleic acid or a vector according to the invention.

Accordingly, particular embodiments of the invention relate torecombinant micro-organisms comprising a polynucleic acid encoding afusion protein consisting of a polypeptide sequence which comprises,more particularly in this order: (1) at least one signal peptide; (2) acarrier domain comprising at least one carbohydrate binding module(CBM), of the type of CBM of a cellulosomal scaffolding protein, fusedto at least one X module of a cellulosomal scaffolding protein; (3) atleast one peptide linker for linking the carrier domain to a polypeptideof interest; and (4) at least one polypeptide of interest. Themicro-organisms of the invention are characterized in thatthey secretethe polypeptide of interest.

In further particular embodiments, micro-organisms are provided whereinthe polynucleic acid encodes a polypeptide sequence which comprises twoor more X modules.

In particular embodiments, micro-organisms are provided wherein thepolypeptide sequence comprises a signal peptide, which is a signalpeptide of a cellulosomal scaffolding protein. Most particularly, thesignal peptide is the signal peptide of the CipC scaffolding protein ofC. cellulolyticum, or the signal peptide of the CipA scaffolding proteinof C. acetobutylicum.

In particular embodiments, micro-organisms are provided wherein thepolypeptide sequence comprises at least one carbohydrate binding modulewhich is a carbohydrate binding module of type-3 a (CBM3a).

In particular embodiments, micro-organisms are provided wherein thepolypeptide sequence comprises at least one X module which is the X2module of the CipC scaffolding protein of C. cellulolyticum, or the X2module of the CipA scaffolding protein of C. acetobutylicum.

More particularly, host cells provided according to the presentinvention are gram-positive bacteria, more particularly members of theclass Clostridia. In further particular embodiments, micro-organismsaccording to the invention are micro-organisms from a Clostridium strainselected from the group comprising C. acetobutylicum and C.beijerinckii.

Micro-organisms according to the invention may comprise one or morenucleic acids, wherein each nucleic acid comprises a sequence encodingone or more polypeptides of interest.

In still another aspect, the invention provides a fusion protein encodedby the polynucleic acid of the invention. In addition the invention alsoprovides a fusion protein which is fused to the signal peptide asdefined herein.

The present invention further relates to a method for the production andsecretion by a host cell, more particularly a bacterial host cell, evenmore particularly a Clostridium host cell, most particularly anon-cellulolytic Clostridium host cell, of at least one heterologous orhomologous polypeptide of interest in a biologically active formcomprising introducing into said host cell of a polynucleic acid or avector according to the invention under conditions effective to causeexpression of the encoded fusion protein, wherein the encoded fusionprotein is secreted by the host cell into the environment of said hostcell. During secretion the signal peptide is optionally cleaved from thefusion protein. Optionally, the polypeptide of interest issimultaneously or additionally cleaved from the carrier domain.

Accordingly, in particular embodiments, the invention provides, methodsfor the production and secretion by a recombinant micro-organism of atleast one heterologous or homologous polypeptide of interest comprisingintroducing into the micro-organism a polynucleic acid encoding a fusionprotein consisting of a polypeptide sequence which comprises, moreparticularly in this order (1) at least one signal peptide; (2) acarrier domain comprising at least one carbohydrate binding module (CBM)of the type of a cellulosomal scaffolding protein, fused to at least oneX module of a cellulosomal scaffolding protein; (3) at least one peptidelinker for linking the carrier domain to a polypeptide of interest; and(4) at least one polypeptide of interest, under conditions effective tocause expression of the encoded fusion protein, wherein the encodedfusion protein is secreted by the recombinant micro-organism into theenvironment of the recombinant micro-organism.

A further aspect of the invention encompasses the use of a polynucleicacid, a vector or a host cell according to the invention for theproduction and secretion of a polypeptide of interest in a biologicallyactive form.

In particular embodiments of the different aspects of the invention, thepolypeptides of interest comprise an enzyme such as a plant cell walldegrading enzyme, and preferably a cellulase. Most particularly, theenzyme is a cellulase of C. cellulolyticum, such as Cel48F or Cel9G.Additionally or alternatively, the polypeptide of interest comprises acellulase CelH of S. degradans strain 2-40.

In another embodiment, polypeptides of interest according to theinvention may comprise a therapeutic protein. Such a therapeutic proteincan be but is not limited to a protein selected from the groupcomprising therapeutic enzymes, cytokines, and antibodies, andpreferably cytokines such as IL-2 or TNFα.

In yet another aspect, the invention relates to a pharmaceuticalcomposition for the treatment of cancer comprising one of a polynucleicacid, a fusion protein, a vector, or a host cell according to theinvention and at least one pharmaceutically acceptable carrier. Moreparticularly, the pharmaceutical composition comprises a host cell, mostparticularly a Clostridium host cell, expressing the polynucleic acidaccording to the invention

The invention further relates to a polynucleic acid, a fusion protein, avector, or a host cell according to the invention for use as amedicament.

In addition, the invention is directed to a polynucleic acid, a fusionprotein, a vector, or a host cell according to the invention fortreating cancer.

In a further aspect, the invention provides methods of treating cancerin a subject in need thereof comprising administering a polynucleicacid, a vector, a host cell or a pharmaceutical composition according tothe invention to said subject, and preferably comprising injecting saidpolynucleic acid, a vector, a host cell or a pharmaceutical compositionat a tumor site in said subject. More particularly, the inventionprovides a method of treating cancer in a subject in need thereofcomprising administering a host cell expressing the polynucleic acidaccording to the invention to said subject. Optionally, said host cellis injected at the tumor site in said subject.

Additional aspects of the present invention will be apparent in view ofthe detailed description, which follows.

DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic representation of different constructs accordingto particular embodiments of the invention. These constructs comprise apolypeptide of interest (cellulase Cel48F or Cel9G) fused to a carrierdomain and a signal peptide. The carrier domain comprises a carbohydratebinding module (CBM3a) from a cellulosomal scaffolding protein fused toone or two hydrophilic domains (Xc or Xa) originating from a same ordifferent cellulosomal scaffolding protein(s). FIG. 1 further indicatesthe secretion of these constructs by C. acetobutylicum.

FIG. 2 is a schematic representation of different constructs accordingto particular embodiments of the invention. The constructs comprise apolypeptide of interest (cellulase “Cel5H”) fused to a carrier domainand a signal sequence. The carrier domain comprises a carbohydratebinding module (CBM3a) from a cellulosomal scaffolding protein fused toone or two X modules (Xa). Cellulase Cel5H comprises a glycosidehydrolase family 5 domain (“5”), a polyserine linker (“sss”), acarbohydrate-binding module family 6 domain (“6”), a glutamicacid-proline-rich region (“eppv”) and a C-terminal domain identified bythe present inventors as a putative carbohydrate-binding module (“DZ”).

FIG. 3 demonstrates the secretion of wild-type Cel5H and Cel5H fused toa carrier domain, compared to a control strain. The carrier domainencompasses a carbohydrate binding module (CBM3a) from a cellulosomalscaffolding protein fused to two hydrophilic domains (Xa). The activityof the culture supernatant was measured on the soluble substratepara-nitrophenyl-cellobiose.

FIG. 4 demonstrates the activity of different proteins including thefusion proteins according to particular embodiments of the invention oncellulose: activity of proteins comprising Cel9G on crystallinecellulose Avicel compared to wild-type Cel9G. The legend is as in FIG.1.

FIG. 5 demonstrates the activity of different proteins including thefusion proteins according to particular embodiments of the invention ondifferent celluloic substrates; (a) activity of proteins comprisingCel5H on soluble substrate para-nitrophenyl-cellobiose; wild-type Cel5H(full line), fusion with one X module (CBM-Xa-5H; dotted line), fusionprotein with two X modules (CBM-Xa-Xa-5H, dashed line) (b) activity ofproteins comprising cel5H on crystalline cellulose Avicel.

DETAILED DESCRIPTION OF THE INVENTION

1. General Definitions

As used herein, the singular forms “a”, “an”, and “the” include bothsingular and plural referents unless the context clearly dictatesotherwise. By way of example, “a cell” refers to one or more than onecells.

The terms “comprising”, “comprises” and “comprised of” as used hereinare synonymous with “including”, “includes” or “containing”, “contains”,and are inclusive or open-ended and do not exclude additional,non-recited members, elements or method steps.

The term “about” as used herein when referring to a measurable valuesuch as a parameter, an amount, a temporal duration, and the like, ismeant to encompass variations of +/−20% or less, preferably +/−10% orless, more preferably +/−5% or less, even more preferably +/−1% or less,and still more preferably +/−0.1% or less from the specified value,insofar such variations are appropriate to perform in the disclosedinvention.

The recitation of numerical ranges by endpoints includes all numbers andfractions subsumed within that range, as well as the recited endpoints.

All documents cited in the present specification are hereby incorporatedby reference in their entirety. In particular, the teachings of alldocuments herein specifically referred to are incorporated by reference.

The present invention is in general directed to polynucleic acids,constructs, molecules and methods for the production and secretion ofpolypeptides by host cells.

In this context the term “secretion” refers to the extracellulardelivery of a polypeptide of interest, i.e. delivery outside a hostcell. In particular this means that the polypeptide of interest isreleased in or accumulates outside the host cell, and for instance inthe “environment” wherein said host cell in grown or is present. In thesame context, translocation refers to the delivery of a polypeptide ofinterest into the periplasmic space.

The terms “polypeptide” and “protein” are used interchangeably hereinand generally refer to a polymer of amino acid residues linked bypeptide bonds, and are not limited to a minimum length of the product.Thus, peptides, oligopeptides, polypeptides, dimers (hetero- and homo-),multimers (hetero- and homo-), and the like, are included within thedefinition. Both full-length proteins and fragments thereof areencompassed by the definition. The terms also include post-expressionmodifications of the polypeptide, for example, glycosylation,acetylation, phosphorylation, etc. Furthermore, for purposes of thepresent invention, the terms also refer to such when includingmodifications, such as deletions, additions and substitutions (e.g.,conservative in nature), to the sequence of a native protein orpolypeptide.

The term “peptide” as used herein preferably refers to a polypeptide asused herein consisting essentially of ≦50 amino acids, e.g., ≦45 aminoacids, preferably ≦40 amino acids, e.g., ≦35 amino acids, morepreferably ≦30 consecutive amino acids, e.g., ≦25, ≦20, ≦15, ≦10 or ≦5amino acids.

As used herein, the term “heterologous polypeptide” refers to apolypeptide that does not naturally occur in a host cell. The term“homologous polypeptide” refers to a polypeptide native or naturallyoccurring in a host cell. In one embodiment, the invention includes hostcells producing the homologous polypeptide via recombinant DNAtechnology. A recombinant protein refers to any protein encoded by apolynucleic acid which has been introduced into the host.

The terms “polynucleic acid” and “nucleic acid” are used interchangeablyherein and generally refer to a polymer of any length composedessentially of nucleotides, e.g., deoxyribonucleotides and/orribonucleotides. Nucleic acids can comprise purine and/or pyrimidinebases, and/or other natural, chemically or biochemically modified (e.g.,methylated), non-natural, or derivatised nucleotide bases. The backboneof nucleic acids can comprise sugars and phosphate groups, as cantypically be found in RNA or DNA, and/or one or more modified orsubstituted (such as, 2′-O-alkylated, e.g., 2′-O-methylated or2′-O-ethylated; or 2′-O,4′-C-alkynelated, e.g., 2′-O,4′-C-ethylated)sugars or one or more modified or substituted phosphate groups. Forexample, backbone analogues in nucleic acids may include phosphodiester,phosphorothioate, phosphorodithioate, methylphosphonate,phosphoramidate, alkyl phosphotriester, sulfamate, 3′-thioacetal,methylene (methylimino), 3′-N-carbamate, morpholino carbamate, andpeptide nucleic acids (PNAs).

The term “polynucleic acid” further specifically encompasses DNA, RNAand DNA/RNA hybrid molecules, specifically including hnRNA, pre-mRNA,mRNA, cDNA, genomic DNA, gene, amplification products, oligonucleotides,and synthetic (e.g. chemically synthesised) DNA, RNA or DNA/RNA hybrids.The terms “ribonucleic acid” and “RNA” as used herein mean a polymer ofany length composed of ribonucleotides. The terms “deoxyribonucleicacid” and “DNA” as used herein mean a polymer of any length composed ofdeoxyribonucleotides. The term “DNA/RNA hybrid” as used herein mean apolymer of any length composed of one or more deoxyribonucleotides andone or more ribonucleotides.

A nucleic acid can be naturally occurring, e.g., present in or isolatedfrom nature, can be recombinant, i.e., produced by recombinant DNAtechnology, and/or can be, partly or entirely, chemically orbiochemically synthesized. A nucleic acid can be double-stranded, partlydouble stranded, or single-stranded. Where single-stranded, the nucleicacid can be the sense strand or the antisense strand. In addition,nucleic acid can be circular or linear.

The term “oligonucleotide” as used herein denotes single strandednucleic acids (nucleotide multimers) of greater than 2 nucleotides inlength and preferably up to 200 nucleotides in length, more preferablyfrom about 10 to about 100 nucleotides in length, even more preferablyfrom about 12 to about 50 nucleotides in length. Oligonucleotides can besynthesised by any method known in the art, e.g., by chemical orbiochemical synthesis, e.g., solid phase phosphoramidite chemicalsynthesis, or by in vitro or in vivo expression from recombinant nucleicacid molecules, e.g., bacterial or retroviral vectors.

As used herein, a “recombinant nucleic acid” is a molecule where thenucleic acid molecule which encodes a polypeptide of interest has beenmodified in vitro, so that its sequence is not naturally occurring, orcorresponds to naturally occurring sequences that are not positioned asthey would be positioned in a genome which has not been modified.

The term “signal sequence” or “secretory signal sequence” or “secretorysignal peptide” or “signal peptide” denote a polypeptide that as acomponent of a larger polypeptide, directs the larger polypeptidethrough a secretory pathway of a host cell in which it is synthesized.The larger polypeptide is commonly cleaved to remove the secretorysignal peptide during transit through the secretory pathway. Thus, whena signal peptide is fused to the amino-terminal end of a heterologousprotein, it directs the protein to the secretory machinery of the hostcell. The heterologous protein is then translocated across the membraneand a specific protease, sometimes referred to as “signal peptidase,”removes the signal peptide and releases the protein, in the present casea fusion protein according to the invention.

The term “cellulosomal scaffolding protein” or “scaffoldin” as usedherein is intended to refer to a scaffolding protein comprised in acellulosome. “Cellulosomes” are extracellular multi-enzymatic complexesthat are present in some cellulolytic micro-organisms and containmultiple copies of enzymes required to break down carbohydrates. Inparticular, cellulosomes are composed of a scaffolding protein, which isattached to various cellulases, hemicellulases, and pectinases, thatwork synergistically to degrade complex cell-wall molecules and thiscomplex allows the organisms to degrade plant cell walls veryefficiently. The scaffolding proteins bring together the various otherproteins in a signaling pathway and allows for their interaction.

The term “protease cleavage site” or “protease target sequence” which iscomprised within the sequence of the polypeptide linker as definedherein, refers to an amino acid sequence that can be recognized byspecific proteases. Cleavage at this site results in the release of thepolypeptide of interest. It should be noted that the linker polypeptidecan be any synthetic polypeptide containing a protease cleavage site, solong as cleavage at this site results in removal of the remainingdomains from the polypeptide of interest. Suitable protease targetsequences which can be used in polynucleic acids encoding fusionproteins as described herein include but are not limited to sequenceswhich can be recognized by serine proteases such as plasmin, thrombin,factor Xa, or trypsin.

As used herein the term “carrier domain” or “carrier module” is intendedto refer to a polypeptide sequence to which a functional domain can befused in accordance with the present invention. The term “functionaldomain” or “functional module” is used herein to refer to a polypeptidesequence comprising a polypeptide of interest which is to be producedand secreted in accordance with the present invention. Fusion betweensaid carrier domain or and said functional domain may be effected bymeans of a linker module.

The expression “at least one” in the context of the present inventionmeans at least two, at least three, at least four, at least five, atleast six, etc. and up to at least ten, and also includes one.

2. Nucleotide Sequences

In a first aspect, the invention relates to a polynucleic acid encodinga fusion protein for facilitating the production and secretion of apolypeptide of interest by a host cell, preferably a bacterial hostcell, as well as various uses thereof. Specifically, it has been foundthat a polynucleic acid encoding a fusion protein as defined hereinallows for the efficient production of polypeptides by a host cell andthe extracellular delivery thereof.

In particular embodiments, the invention provides a polynucleic acidencoding a fusion protein consisting of a polypeptide sequence whichcomprises:

-   -   a carrier domain comprising at least one carbohydrate binding        module (CBM) of a cellulosomal scaffolding protein fused to at        least one X module or hydrophilic module of a cellulosomal        scaffolding protein;    -   at least one polypeptide of interest, and    -   at least one peptide linker for linking the carrier domain to        the polypeptide of interest.

In further particular embodiments, the invention provides a polynucleicacid encoding a fusion protein consisting of a polypeptide sequencewhich comprises:

-   -   a suitable secretion signal peptide    -   a carrier domain comprising at least one carbohydrate binding        module (CBM) of a cellulosomal scaffolding protein fused to at        least one X module or hydrophilic module of a cellulosomal        scaffolding protein;    -   at least one polypeptide of interest, and    -   at least one peptide linker for linking the carrier domain to        the polypeptide of interest.

In particular embodiments, the invention provides a polynucleic acidencoding a fusion protein consisting of a polypeptide sequence whichcomprises:

-   -   i) at least one suitable secretion signal peptide    -   ii) at least one carbohydrate binding module (CBM) of a        cellulosomal scaffolding protein,    -   iii) at least one X module or hydrophilic module of a        cellulosomal scaffolding protein, which is fused to said CBM        (i),    -   iv) at least one polypeptide of interest,    -   v) at least one peptide linker for linking the X module or        hydrophilic module (ii) to the polypeptide of interest (iii).

A polynucleic acid encoding a fusion protein as described herein thus inparticular embodiments comprises an in frame nucleic acid secretorysequence for directing the encoded fusion protein out of the host cell.Secretion may thus result in the presence or accumulation of theproduct, i.e. the entire fusion protein (e.g. CBM-X-enzyme) in theenvironment, e.g. a culture medium, comprising the host cell.

Thus, in a more particular embodiment, the invention provides apolynucleic acid encoding a fusion protein consisting of a polypeptidesequence which comprises:

-   -   a suitable secretion signal peptide    -   a carrier domain comprising at least one carbohydrate binding        module (CBM) of a cellulosomal scaffolding protein fused to at        least one X module or hydrophilic module of a cellulosomal        scaffolding protein;    -   at least one peptide linker for linking the carrier domain to a        polypeptide of interest; and    -   at least one polypeptide of interest.

Most particularly, the sequences are arranged in the order they arelisted above.

More particularly, the invention provides a polynucleic acid encoding afusion protein consisting of a polypeptide sequence which comprises:

-   -   i) at least one carbohydrate binding module (CBM) of a        cellulosomal scaffolding protein,    -   ii) at least one X module or hydrophilic module of a        cellulosomal scaffolding protein, which is fused to said CBM        (i),    -   iii) at least one signal peptide e.g. a signal peptide from a        bacterial scaffolding protein    -   iv) at least one polypeptide of interest,    -   v) at least one peptide linker for linking the X module or        hydrophilic module (ii) to the polypeptide of interest (iii).

Accordingly, the invention provides polynucleic acid sequencescomprising individual sequences encoding each of the modules describedabove operably linked, more particularly covalently linked, such thatexpression of the polynucleic acid sequences results in a fusion proteinas described herein.

Suitable nucleic acids sequences of secretory signal sequences which canbe used in polynucleic acids encoding fusion proteins as describedherein are described elsewhere herein and include but are not limited toa signal peptide of a cellulosomal scaffolding protein, such as e.g. thesignal peptide of the CipC scaffolding protein of C. cellulolyticum ATCC35319 (gene bank U40345), or the signal peptide of the CipA scaffoldingprotein of C. acetobutylicum ATCC 824 (gene bank AE007606 or AE001437).

In particular embodiments the fusion protein as defined herein may becleaved during secretion, such that secretion results in the presence oraccumulation of the polypeptide of interest in the environment, e.g. aculture medium, comprising the host cell. To that end, fusion proteinsof the invention can also be engineered to contain a cleavage site toaid in protein recovery. Therefore in particular embodiments theinvention provides a polynucleic acid encoding a fusion protein asdescribed herein having a peptide linker comprising a protease cleavagesite.

3. Fusion Protein

In another aspect, the present invention relates to a fusion proteinthat is encoded by a polynucleic acid according to the invention.

The present fusion protein which may also be denoted as a chimericprotein. “Fusion” refers to the joining together of a polynucleic acidencoding a polypeptide of interest and a polynucleic acid encoding acarrier domain comprising one or more modules, in frame. Expression ofthe joint polynucleic acids results in a chimeric protein also namedhereinafter a “fusion protein”. The fusion protein of the presentinvention may comprise an enzymatic or chemical cleavage site upstreamand preferably adjacent the N-terminus of the polypeptide of interestand/or an enzymatic or chemical cleavage site downstream and preferablyadjacent the C-terminus of the domain provided upstream of thepolypeptide of interest thereby providing a means for recovering thepolypeptide of interest from the fusion protein through use of acleaving agent.

In general, a fusion protein according to the invention consists of apolypeptide sequence, which comprises:

-   -   a carrier domain, which preferably comprises at least one        carbohydrate binding module (CBM) of a cellulosomal scaffolding        protein fused to at least one hydrophilic domain of a        cellulosomal scaffolding protein; and    -   a functional domain which comprises at least one polypeptide of        interest.

More particularly, a fusion protein according to the invention consistsof a polypeptide sequence, which comprises:

-   -   a suitable signal peptide sequence    -   a carrier domain, which preferably comprises at least one        carbohydrate binding module (CBM) of a cellulosomal scaffolding        protein fused to at least one hydrophilic domain of a        cellulosomal scaffolding protein; and    -   a functional domain which comprises at least one polypeptide of        interest.

The carrier domain is linked to the functional domain by means of alinker module.

It shall be further noted that in accordance with particular embodimentsthe present invention, the fusion protein is a protein construct thathas been cleaved from and thus does no longer include a signal peptide.In these embodiments, the signal peptide which is fused to the fusionprotein is cleaved from said fusion protein during secretion.Optionally, in case the polypeptide linker mentioned above containsprotease cleavage site, the polypeptide of interest may be furthercleaved from the remaining part of the fusion protein upon action ofsuitable protease(s), which are able to recognize said protease cleavagesite and to cleave the polypeptide sequence at that site. Accordingly,the term “fusion protein” as used herein may refer to either thepolypeptide sequence as synthesized within the cell (i.e. comprising thesignal sequence) or after secretion, whereby the signal sequence hasoptionally been released or cleaved therefrom.

The object of the production of polypeptides of interest in the form offusion proteins according to the invention is to ensure or increasesecretion of the polypeptide of interest.

In particular embodiments, the fusion protein of the present inventionhas particular improved properties, such as e.g. increased activitycompared to the isolated polypeptide of interest. For instance, inparticular embodiments, for example where the polypeptide of interest isan enzyme, the presence of one or more carbohydrate binding modulesand/or one or more hydrophilic modules in the fusion protein mayincrease the activity of the enzyme compared to the isolated enzyme.Accordingly, in particular embodiments, the invention relates to fusionproteins comprising a carrier domain according to the invention havingimproved properties compared to the polypeptide of interest.

In alternative particular embodiments, the fusion protein of theactivity of the fusion protein is similar or decreased compared to thenative polypeptide.

The separate modules comprised in the present fusion protein and partsthereof such as the carrier domain and the functional domain, as definedherein will be discussed into more detail hereunder.

A. Carbohydrate Binding Module

A first module in a carrier domain, or in a fusion protein according tothe invention comprises a carbohydrate binding module.

The terms “carbohydrate binding module”, “carbohydrate bindingmolecule”; “carbohydrate binding protein” and “carbohydrate bindingdomain” are used herein as synonym and refer to a protein or anessential part, or a homologue thereof, which is capable of binding apolysaccharide substrate, such as e.g. cellulose. The CarbohydrateBinding Modules (CBMs) are functionally independent modules, frequentlyfound in nature associated to proteins involved in biomass breakdown.These modules are defined as sequences of amino acids, present inenzymes which act on carbohydrates, exhibiting tri-dimensional structureand carbohydrate binding ability. The carbohydrate binding modulespreferably include carbohydrate binding modules of a cellulosomalscaffolding proteins.

The term “essential parts thereof” in this context refers to parts ofcarbohydrate binding modules which are capable of binding carbohydrates.

The term “homologue” of a carbohydrate binding protein as used hereinrefers to a protein which has an amino acid sequence that has at least30% identity, preferably at least 40%, 50%, 60%, 70%, 80% or 90%identity, most preferably at least 95% identity with a functionalportion of the amino acid sequence of a carbohydrate binding protein. Itshould be understood that instead of % “identity”, also thecorresponding % “similarity” can be used to define homologues accordingto the invention.

In a particular embodiment, a carrier domain or a fusion proteinaccording to the invention comprises a carbohydrate binding module froma cellulosomal scaffolding protein, as defined above. In furtherparticular embodiments the carrier domain comprises a carbohydratebinding module from an enzyme but which is similar to a carbohydratebinding module from a cellulosomal scaffolding protein (e.g. CBM3b)

In further particular embodiments, a carrier domain or a fusion proteinaccording to the invention comprises a carbohydrate binding module whichis a CBM3 module, i.e. a carbohydrate binding type-3 module.Carbohydrate-binding modules have been classified into more than 40families according to sequence homology. Several cellulolytic enzymesshare a conserved region of about 150 amino acid residues, the CBM3domain. The CBM3 domain has been classified in three different subtypes,termed family IIIa, IIIb and IIIc. In a preferred embodiment, a carrierdomain or a fusion protein according to the invention comprises acarbohydrate binding module which is a CBM3 module of type IIIa or IIIb.In further particular embodiments the carrier domain or a fusion proteinaccording to the invention comprises a carbohydrate binding module whichis a CBM3 module of type IIIa. The carbohydrate binding modules offamily IIIa bind to crystalline cellulose.

Particular examples of carbohydrate binding modules comprised in acarrier domain or a fusion protein according to the invention comprisebut are not limited to carbohydrate binding modules of cellulosomalscaffolding proteins selected from the group comprising cellulosomeintegrating protein A (CipA) of Clostridium thermocellum (gene bankX67406 or X67506), cellulosome integrating protein C (CipC) ofClostridum cellulolyticum (gene bank U40345), cellulose binding proteinA (CbpA) of Clostridum cellulovorans (gene bank M73817), and cellulosomeintegrating protein A (CipA) of Clostridium acetobutylicum (gene bankAE007606 or AE001437).

A particular example of a carbohydrate binding module comprised in acarrier domain or a fusion protein according to the invention is thecarbohydrate binding module of the scaffolding protein CipC ofClostridium cellulolyticum (gene bank U40345). The C. cellulolyticumcellulosome is organized around the scaffolding protein CipC, whichpermits the binding of the different cellulosomal enzymes viainteractions of dockerin-cohesin domains.

In a particular embodiment the carbohydrate binding module compriseshomologues of the carbohydrate binding module of the scaffolding proteinCipC of Clostridium cellulolyticum. Therefore, according to a furtherembodiment, the invention also relates to a carrier domain or a fusionprotein as described above, wherein said at least one carbohydratebinding module comprises a polypeptide having at least 30% identity,preferably at least 40%, 50%, 60%, 70%, 80% or 90% identity, mostpreferably at least 95% identity with the carbohydrate binding module ofthe scaffolding protein CipC of Clostridium cellulolyticum. It should beunderstood that instead of % “identity”, also the corresponding %“similarity” can be used to define homologues according to theinvention.

B. X module

Another module of a fusion protein according to the invention comprisesat least one X module, more particularly a hydrophilic module, andpreferably a hydrophilic module of a cellulosomal scaffolding protein.

The terms “hydrophilic domain”, “hydrophilic module” and “X module” areused herein as synonym and refer to a hydrophilic domain of acellulosomal scaffolding protein. In particular embodiments, theX-module is an X-module of a mesophilic Clostridium cellulosomalscaffolding protein.

Thus in particular embodiments, the X module comprised in a fusionprotein according to the invention is of bacterial origin, preferablyfrom a bacteria of the genus Clostridia, more particularly a mesophilicclostridia, e.g. from Clostridium thermocellum, Clostridiumcellulolyticum, Clostridium acetobutylicum, Clostridium josui orClostridium cellulovorans. It shall be noted that X modules found in C.acetobutylicum, C. cellulolyticum, C. cellulovorans, C. josui may bereferred to as “X2” modules, while X modules found in C. thermocellummay be called “X1” modules.

Particular examples of X modules comprised in a carrier domain or afusion protein according to the invention comprise but are not limitedto hydrophilic domains of cellulosomal scaffolding proteins selectedfrom the group comprising cellulosome integrating protein A (CipA) ofClostridium thermocellum (gene bank X67406 or X67506), cellulosomeintegrating protein C (CipC) of Clostridum cellulolyticum (gene bankU40345), cellulose binding protein A (CbpA) of Clostridum cellulovorans(gene bank M73817), cellulosome integrating protein A (CipA) ofClostridium josui (gene bank AB004845) and cellulosome integratingprotein A (CipA) of Clostridium acetobutylicum (gene bank AE007606 orAE001437).

A particular example of an X module comprised in a carrier domain or afusion protein according to the invention is the X2 module of thescaffolding protein CipC of Clostridium cellulolyticum (gene bankU40345).

Another particularly preferred example of an X module comprised in thefusion protein according to the invention is the X2 module of thescaffolding protein CipA of Clostridium acetobutylicum (gene bankAE007606 or AE001437).

In further embodiments, the X-module is a module homologous to the Xmodules described herein.

In particular embodiments the X module is a homologue of the hydrophilicmodule of the scaffolding protein CipC of Clostridium cellulolyticum orof the scaffolding protein CipA of Clostridium acetobutylicum.Therefore, according to a further embodiment, the invention also relatesto fusion proteins, nucleic acid sequences encoding them and host cells,more particularly recombinant micro-organisms as described above,wherein said at least one X module comprises a polypeptide having atleast 30% identity, preferably at least 40%, 50%, 60%, 70%, 80% or 90%identity, most preferably at least 95% identity with the hydrophilicmodule of the scaffolding protein CipC of Clostridium cellulolyticum orof the scaffolding protein CipA of Clostridium acetobutylicum. It shouldbe understood that instead of % “identity”, also the corresponding %“similarity” can be used to define homologues according to theinvention.

It shall be further noted that in accordance with the present inventionthe CBM module and the X module applied in a carrier domain or a fusionprotein according to the invention may originate from the same or fromdifferent cellulosomal scaffolding proteins.

In one particular embodiment, the present invention relates to apolynucleic acid encoding a fusion protein having a carrier domaincomprising a carbohydrate binding module (CBM) of a cellulosomalscaffolding protein fused to one X module of a same or a differentcellulosomal scaffolding protein. In another particular embodiment, thepresent invention relates to a polynucleic acid encoding a fusionprotein having a carrier domain comprising a carbohydrate binding module(CBM) of a cellulosomal scaffolding protein fused to two of the same orto two different X modules of a same or a different cellulosomalscaffolding protein.

In particular embodiments the fusion protein comprises two X modules,three X modules, or four or more X modules. These may be located in thefusion protein adjacent to each other or separated by one or more othermodules of the fusion protein.

C. Signal Peptide

In particular embodiments, the fusion protein, comprising a carrierdomain ensuring the secretion of the polypeptide of interest accordingto the present invention, further comprises a sequence encoding asecretion signal sequence. In the constructs according to theseembodiments of invention, this secretion signal sequences is linked toone of the other sequences of the construct, i.e. either the sequenceencoding the CBM domain, the X module or the sequence encoding thepolypeptide of interest, such that the signal sequence and thepolypeptide of interest are operably linked, more particularlycovalently linked. In this connection, “operably linked” denotes thatthe sequence encoding the signal sequence and the sequence encoding thepolypeptide to be secreted are connected in frame or in phase, such thatupon expression the signal peptide facilitates the secretion of thepolypeptide so-linked thereto.

It shall be appreciated that suitable signal sequences may depend on thetype of micro-organism in which secretion is desired. For example,distinct signal sequences may be required in different Gram-positivebacteria. By means of example and not limitation, secretion inGram-positive bacteria, and in particular in Clostridium such as C.acetobutylicum, may be achieved using the signal sequence of the Cel5Aprecursor polypeptide of C. cellulolyticum (exemplary sequence: Genbankacc. no. AAA51444, seq version 1 revised on Oct. 31, 1994), or of theCipC precursor scaffolding protein of C. cellulolyticum (exemplarysequence: Genbank acc. no. AAC28899, seq. version 2 revised on Dec. 5,2005), or of the CipA precursor scaffolding protein of C. acetobutylicum(exemplary sequence: Genbank acc. no. AAK78886, seq. version 1 revisedon Jan. 19, 2006).

It shall also be appreciated that native (or homologous, endogenous)signal peptides of polypeptides to be expressed by the micro-organismsas taught herein may be employed, insofar as they are functional in saidmicro-organisms. Hence, by means of example, secretion of Cel48F orCel9G of Clostridium Cellulolyticum may be achieved using the CipCscaffolding protein of C. cellulolyticum. Similarly, secretion of Cel5Hor related polypeptides in a heterologous organism may be achieved usingthe endogenous or homologous secretion signal sequence of the Cel5Hprecursor polypeptide.

D. Polypeptide of Interest

The present fusion protein or a functional domain thereof according tothe invention further comprises a protein of interest to be produced andsecreted by constructs and methods as provided herein. The protein whichis produced and secreted by a—preferably bacterial—host as definedherein can be any protein of interest. In a preferred embodiment, it isa heterologous protein, i.e. heterologous to the host. Alternatively,the protein is homologous.

The terms “polypeptide of interest” and “protein of interest” are usedherein as synonym and refer to a protein that is produced and secretedby a host cell as defined herein.

The present invention is not limited in the type or function ofpolypeptide of interest that can be produced and secreted in accordancewith the present invention. The nature of the protein of interest isdetermined by the application of the nucleic acids and host cellscomprising them.

In one embodiment, said polypeptide of interest is an enzyme, preferablyselected from the group comprising but not limited to proteases,reductases, lipases, kinases, phophatases, oxidases, and carbohydrases.

For example, said polypeptide of interest is an enzyme selected from thegroup comprising but not limited to transferases (EC.2), isomerases(EC.5), oxidoreductases (EC.1) comprising but not limited to enzymes ofgroup EC 1.10.3 including laccase or peroxidases (EC 1.11.1) includingligninase and lignin peroxidase, and hydrolases (EC.3) comprising butnot limited to carboxylic ester hydrolases (EC 3.1.1) includinghemicellulase, and glycosidases (EC 3.2.1) including endoglucanases,exoglucanases, alpha-amylase, glucoamylase, pectinase, endo-glucosidaseH, cellulase, cellobiohydrolase, and endo-processive cellulase.

In particular embodiments said polypeptide of interest is a plant cellwall degrading enzyme. The secretion of such enzymes by micro-organismsis of interest in the context of degradation of plant material, e.g. inthe context of biofuel production. The term “plant cell wall degradingenzymes” is used herein to refer to enzymes which catalyze the cleavageof cellulosic or lignocellulosic materials, and include but are notlimited to cellulases, hemicellulases, laccases, cellobiohydrolases andother enzymes involved in breaking down cellulose and hemicellulose intosimple sugars such as glucose, xylose, arabinose, mannose and galactose.

In a particular embodiment said polypeptide is a cellulase. This termincludes processive and non-processive cellulases. Processive cellulasewill continue to interact with a single polysaccharide strand,non-processive cellulase will interact once then disengage and engageanother polysaccharide strand. Explicitly, but not exclusively, includedwithin the term cellulases are those enzymes which fall under the EnzymeClassification heading EC 3.2.1.4 enzymes, also calledβ-1,4-endoglucanases, cleave β-1,4-glycosidic linkages randomly alongthe cellulose chain, EC 3.2.1.91 enzymes also called cellobiohydrolasesor exoglucanases which sequentially release cellobiose or glucose fromone extremity of the cellulose chain, and EC 3.2.1.4/EC 3.2.1.91 enzymesalso called endo-processive cellulases which display a mixed mode ofaction (both endo and exo glucanase).

According to particular embodiments, the plant cell wall degradingenzymes used in the present invention are of microbial origin, e.g. offungal or bacterial origin, preferably of bacterial origin, for examplefrom a bacterium of the genus Alteromonadaceae, e.g. Saccharophagusdegradans strain 2-40, of the genus Thermomonospora, e.g., from T.fusca, of the genus Cellulomonadaceae e.g. C. fimi, of the genusClostridia, e.g. from Clostridium thermocellum, Clostridiumcellulolyticum, Clostridium acetobutylicum, Clostridium cellulovorans,Clostridium josui. In another embodiment, plant cell wall degradingenzymes used in the present invention are of fungal origin, for examplefrom a fungus of the genus Neocallimastigomycota, e.g. from N.patriciarum or Orpinomyces sp strain PC-2.

Particular examples of cellulases suitable for use in a fusion proteinor a functional domain thereof according to the invention include butare not limited to:

-   -   cellulases of C. cellulolyticum selected from the group        comprising Cel48F, Cel9G, Cel9R, Cel9P, Cel9E, Cel9H, Cel9J,        Cel9M, Cel8C, Cel5N, and Cel5A;    -   cellulases of C. thermocellum selected from the group comprising        Cel9D, Cel9J, CBH9A, Cel9H, Cel9K, Cel5E, Cel48S, Cel9F, Cel9N,        Cel9Q, Cel5O, Cel5B, Cel5G, Cel8A, Cel5C and Cel91;    -   cellulases of C. acetobutylicum selected from the group        comprising Cel48A, Cel9G, Cel9R, Cel9P, Cel9E, Cel9H, Cel9J,        Cel9M, and Cel5A;    -   cellulases of S. degradans strain 2-40 selected from the group        comprising Cel9A, Cel9B, Cel5J, Cel51, Cel5F, Cel5H, Cel5D,        Cel5B, Cel9G, Cel5E, Cel5A, Cel5C and Cel6A.    -   putative cellulases from Pseudomonas species ND 137 such as        Acla.

In further particular examples, a cellulase suitable for use in a fusionprotein or a functional domain thereof according to the inventionincludes the cellulases Cel48F or Cel9G of C. cellulolyticum.

In further particular examples, a cellulase suitable for use in a fusionprotein or a functional domain thereof according to the inventionincludes the cellulase Cel5H of S. degradans strain 2-40.

In further particular embodiments, the polypeptide of interest to beproduced and secreted in accordance with the present invention is atherapeutic protein. A “therapeutic protein” as used herein, refers aprotein, peptide, glycoprotein or glycopeptide that can be administeredto a subject to treat a disease or dysfunction or to improve health ofthe subject. It includes both molecules which in themselves exert atherapeutic effect and molecules which act on or combine with anothermolecule to exert a therapeutic effect, such as part of a combinationdrug or a pro-drug converting enzyme. In particular embodiments thesubject is an animal or a human. In a further preferred embodiment, thetherapeutic protein is a human protein or an animal protein, e.g. from arodent, e.g. rat, mice. In another further particular embodiment, thedisease or dysfunction includes a cancer. Accordingly, in particularembodiments, the polypeptide of interest is an anti-tumor agent.

In particular embodiments the therapeutic protein is an active protein,e.g., has enzymatic activity, or biological activity, such as bindingactivity to a ligand or receptor, ability to activate an intracellularsignal transduction pathway, or ability to elicit an immune response ina mammal, e.g., a human. The therapeutic protein may be glycosylated orotherwise modified in vitro by one or more glycosyltransferases.

In particular embodiments the protein of interest for use in a fusionprotein or a functional domain thereof according to the invention is atherapeutic protein selected from the group comprising therapeuticenzymes, cytokines, and antibodies (including all known forms ofantigen-binding molecules). It shall be noted that in accordance withthe present invention the term antibodies also includes “catalyticantibodies”.

In a further particular embodiment, a therapeutic protein is selectedfrom the group comprising cytokines such as but not limited to IL-2,IL-12, GM-CSF (granulocyte-macrophage colony-stimulating factor), TNF(tumor necrosis factor)-α, etc.

The use of therapeutic proteins is more particularly envisaged for thetherapeutic applications of the invention described herein.

In further particular embodiments, the polypeptide of interest is adiagnostic polypeptide, such as an antibody. These may be of interest inthe diagnostic use of the host cells of the present invention.

E. Linker Module

A fusion protein according to the invention typically comprises anothermodule, which consists of at least one peptide linker for linking thecarrier domain of the protein to the functional domain or thepolypeptide of interest.

Preferably the peptide sequence, linking the carbohydrate binding moduleto the hydrophilic module is a sequence which is known in the art andwhich can be conveniently found in cellulosomal scaffolding proteins.

Said linker polypeptide preferably comprises a polypeptide of at least3, preferably at least 4 or 5, most preferably at least 7, and morepreferably at least 12 amino acids. Preferably said linker is apolypeptide comprising between 3 and 15 amino acids. Preferably saidlinker is a polypeptide comprising non-charged amino acids such asglycine, serine, cysteine, asparagine, tyrosine, glutamine, alanine,valine, proline, threonine, and preferably glycine or serine.

Suitable examples of linker polypeptides comprise linker polypeptidesfound in bacterial cellulosomal scaffolding proteins such as but notlimited to CipA of C. acetobutylicum (AE007606), CipC of C.cellulolyticum (U40345), CipA of C. thermocellum (X67406 or X67506),CbpA of Clostridum cellulovorans (M73817), CipA of Clostridium josui(AB004845).

As mentioned above, a peptide linker as defined herein may comprise aprotease cleavage site. Cleavage at this site results in the release ofthe polypeptide of interest.

4. Vectors

According to a further aspect of the present invention, there areprovided expression constructs to facilitate introduction into a hostcell and preferably a bacterial cell and/or facilitate expression and/orfacilitate maintenance of the polynucleotide sequence encoding a fusionprotein according to the invention. The expression constructs may beinserted into a plasmid or a vector, which may be commerciallyavailable.

By “vector” is meant a polynucleotide molecule, preferably a DNAmolecule derived, for example, from a plasmid, bacteriophage, or plantvirus, into which a polynucleotide can be inserted or cloned. A vectorpreferably contains one or more unique restriction sites and can becapable of autonomous replication in a defined host cell, or beintegrable with the genome of the defined host such that the clonedsequence is reproducible. The choice of the vector will typically dependon the compatibility of the vector with the host cell into which thevector is to be introduced.

According to an embodiment of the present invention, the expressionconstruct is an expression vector, suitable for transformation into hostorganisms, preferably bacteria, and suitable for maintenance andexpression of a fusion protein according to the present invention in atransformed host cell.

An “expression vector” is a construct that can be used to transform aselected host cell and provides for expression of a coding sequence inthe selected host. Expression vectors can for instance be cloningvectors, binary vectors or integrating vectors. The invention thus alsorelates to a vector comprising any of the nucleic acids described above.Said vector may further comprise regulatory sequences for controllingexpression of the nucleic acid in said host cell. Particularly useful inthe practice of this invention are expression vectors that provide forthe expression of bacterial cells of nucleic acid encoding a fusionprotein as defined herein. In general, expression involves the use of anexpression vector that is able to replicate efficiently in a host cell,such that the host cell accumulates many copies of the expression vectorand, in turn, synthesizes high levels of a desired product (fusionprotein) encoded by the expression vector.

The terms “regulatory sequences” and “control sequence” used herein areto be taken in a broad context and refer to regulatory nucleic acidsequences capable of driving and/or regulating expression of thesequences to which they are ligated (covalently linked) and/or operably,linked. The control sequences differ depending upon the intended hostorganism and upon the nature of the sequence to be expressed. Forexpression of a protein in prokaryotes, the control sequences generallyinclude a promoter, a ribosomal binding site, and a terminator. Ineukaryotes, control sequences generally include promoters, terminatorsand, in some instances, enhancers, and/or 5′ and 3′ untranslatedsequences. The term ‘control sequence’ is intended to include, at aminimum, all components necessary for expression, and may also includeadditional advantageous components. According to a preferred embodimentof the present invention, the control sequence is operable in abacterium, and preferably a gram positive bacterium; preferably thecontrol sequence is a sequence derived from a gram positive bacterium.The term “control sequence” encompasses a promoter or a sequence capableof activating or enhancing expression of a nucleic acid molecule in ahost cell.

According to one embodiment of the present invention, the expressionconstruct is a bacterial expression vector, suitable for transformationinto bacteria and suitable for maintenance and expression of a fusionprotein according to the present invention in a transformed bacterialcell. The invention thus also relates to a vector comprising any of thenucleic acids described above. Said vector may further compriseregulatory sequences for controlling expression of the nucleic acid in abacterial cell.

Expression and cloning vectors usually contain a promoter that isrecognized by the host organism and is (covalently and) operably linkedto the nucleic acid encoding the polypeptide of interest. Promoters areuntranslated sequences located upstream (5′) to the start codon of astructural gene (generally within about 100 to 1000 bp) that control thetranscription and translation of a particular nucleic acid sequence,such as that encoding a fusion protein as defined herein, to which theyare operably linked. Such promoters typically fall into two classes,inducible and constitutive. Inducible promoters are promoters thatinitiate increased levels of transcription from nucleic acid under theircontrol in response to some change in culture conditions, e.g., thepresence or absence of a nutrient or a change in temperature. At thistime, a large number of promoters recognized by a variety of potentialhost cells are well known. These promoters are operably linked tonucleic acid encoding the polypeptide of interest by removing thepromoter from the source nucleic acid by restriction enzyme digestionand inserting the isolated promoter sequence into the vector. Both thenaturally occurring promoter sequence and many heterologous promotersmay be used to direct amplification and/or expression of the polypeptideof interest. In general, plasmid vectors containing promoters andcontrol sequences that are derived from species compatible with the hostcell are used with these hosts. The vector ordinarily carries one ormore replication sites as well as marker sequences, which are capable ofproviding phenotypic selection in transformed cells.

Promoters suitable for use with prokaryotic hosts illustratively includethe β-lactamase and lactose promoter systems, alkaline phosphatase, thetryptophan (trp) promoter system and hybrid promoters such as the tacpromoter. However, other functional bacterial promoters are suitable.Their nucleotide sequences are generally known, thereby enabling askilled worker operably to ligate them to nucleic acid encoding theprotein secretion molecule as defined herein using linkers or adaptersto supply any required restriction sites. Promoters for use in bacterialsystems. A Shine-Dalgarno sequence should also be operably linked to thenucleic acid encoding the protein secretion molecule as defined herein.

According to one embodiment of the invention, the vectors comprise aconstitutive promoter. Examples of constitutive promoters suitable forthe constructs and methods according to the present invention includebut are not limited to the CaMV35S promoter, GOS2, actin promoter,ubiquitin promoter, thiolase promoter.

According to another embodiment of the invention, the vectors comprisean inducible promoter. Examples of inducible promoters suitable for theconstructs and methods according to the present invention include butare not limited to the lac promoter or xylose inducible promoter

Optionally, the present expression vectors will also contain sequencesnecessary for the termination of transcription and for stabilizing themRNA, and may thus contain one or more transcription terminationsequences. The term “transcription termination sequence” encompasses acontrol sequence at the end of a transcriptional unit, which signals 3′processing and termination of transcription. Additional regulatoryelements, such as transcriptional or translational enhancers, may beincorporated in the expression construct.

The expression constructs of the invention may further include an originof replication that is required for maintenance and/or replication in aspecific cell type. One example is when an expression construct isrequired to be maintained in a bacterial cell as an episomal geneticelement (e.g. plasmid or cosmid molecule). Preferred origins ofreplication include, but are not limited to the f1-ori, colE1 ori, andGram+ bacteria origins of replication.

The expression construct may optionally comprise a selectable markergene. As used herein, the term “selectable marker gene” includes anygene, which confers a phenotype on a cell in which it is expressed tofacilitate the identification and/or selection of cells which aretransfected or transformed with an expression construct of theinvention. Suitable markers may be selected from markers that conferantibiotic or herbicide resistance or visual markers. Examples ofselectable marker genes include genes encoding neomycinphosphotransferase (nptII), hygromycin phosphotransferase (hpt) orBasta. Further examples of suitable selectable marker genes includeresistance genes against ampicillin (AmpR), tetracydine (TcR), kanamycin(KanR), phosphinothricin, and chloramphenicol or thiamphenicol (CAT).Other suitable marker genes provide a metabolic trait, for example manA.Visual marker genes may also be used and include for examplebeta-glucuronidase (GUS), luciferase and Green Fluorescent Protein(GFP).

Construction of suitable vectors containing one or more of the abovelisted components and including the desired coding and control sequencesemploys standard ligation techniques. Isolated plasmids or nucleic acidfragments are cleaved, tailored, and religated in the form desired togenerate the plasmids required.

5. Host Cells

According to one aspect, the present invention relates to a host cellcomprising a polynucleic acid or a vector as defined herein.

The term “host cell” refers to those cells capable of growth in cultureand capable of expressing a polynucleic acid as defined herein and thuscapable of producing and secreting a fusion protein as defined herein.The host cells of the present invention encompass in vitro cell culturesand include prokaryotic cells. Particular embodiments relate tomicro-organisms. Particular examples of host cells which may be used inaccordance with the present invention include bacterial cells.

It shall be noted that the term “host cell” is intended to include allforms of the life cycle of the host cell such as spores.

In particular embodiments, the host cells envisaged in the context ofthe invention are bacterial cells, in particular gram positive bacterialcells. The term “Gram-positive bacteria” is intended to include theart-recognized definition of this term. Gram-positive bacteria include,but are not limited to, Bacillus, Geobacillus, Clostridium,Streptococcus, Cellulomonas, Corynebacterium, Lactobacillis,Lactococcus, Oenococcus and Eubacterium.

According to a particular embodiment of the present invention, said hostcell is a gram-positive bacterial cell member of the class Clostridia,more preferably a member of the genus Clostridium.

Most particularly, Clostridia strains are envisaged which are amenableto genetic manipulation such as but not limited to C. acetobutylicum, C.sporogenes, C. beijerinckii etc.

The selection of the host cell may be determined by the envisagedapplication. Most particularly, the invention is applicable to strainsfor which secretion of polypeptides of interest is problematic.

In one embodiment said host cell is a member of the group comprisingsolventogenic, i.e. solvent producing, Clostridia strains. Particularlypreferred host cells according to this embodiment of the invention aresolvent-producing Clostridia strains selected from the group comprisingC. acetobutylicum, for instance C. acetobutylicum strain ATCC824, and C.beijerinckii, for instance C. beijerinckii strain ATCC17778.

In another embodiment said host cell is a member of the group comprisingsporogenic bacteria, such as but not limited to bacteria of the genusBacillus, Clostridium (more particularly for therapeutic applications,where administration of spores is of interest). Particularly preferredhost cells according to this embodiment of the invention are Clostridiastrains selected from the group comprising C. sporogenes, for instanceC. sporogenes strain DSM767, and C. acetobutylicum, for instance C.acetobutylicum strain ATCC824.

The polynucleic acid molecules or vectors according to the invention mayeither be integrated into the genome of the host cell or it may bemaintained in some form extrachromosomally.

More in particular, host cells may be transformed with the expressionvectors of this invention and cultured in conventional nutrient mediamodified as is appropriate for inducing promoters, selectingtransformants or amplifying genes. “Transformation” means introducingnucleic acid into an organism so that the nucleic acid is replicable,either as an extrachromosomal element or by chromosomal integration.Methods used herein for transformation of the host cells are well knownto a skilled person. The culture conditions, such as temperature, pH andthe like, are those previously used with the host cell selected forexpression, and will be apparent to the ordinarily skilled artisan.

6. Methods for Producing and Secreting Polypeptides

In another aspect, the present application is directed to a method forthe production and secretion by a host cell, preferably a bacterial hostcell, of at least one heterologous or homologous polypeptide of interestin a biologically active form comprising introducing into said host cellof a polynucleic acid or a vector according to the invention underconditions effective to cause expression of the encoded fusion protein,wherein the encoded fusion protein is secreted by the host cell into theenvironment of said host cell.

During secretion a signal peptide is preferably cleaved from said fusionprotein such that the fusion protein is released in the host environment(e.g. a culture medium). In another aspect, a protease target sequenceintroduced in the linker connecting the carrier domain to the functionaldomain as defined herein, and the protein of interest is cleaved byprotease(s) to release in the host environment the protein of interestcleaved from the remaining fusion protein.

Preferably said host cell is a bacterial host cell as defined above.

The environment of said host cell is intended to refer to the placewherein said bacterium in grown. In one embodiment the environment ofsaid bacterium may be a culture medium wherein said bacterium is grown.In another embodiment the environment of said bacterium may be a tissueof a living being, e.g. a human or animal tissue, in particular in thecase of therapeutic applications contemplated in the present invention.

The present invention also relates to the use of a polynucleic acid, avector or a host cell according to the invention, for the production andsecretion of a polypeptide of interest in a biologically active form.

The invention further relates to the use of a carrier domain as definedherein for controlling the secretion of a polypeptide of interest,preferably a polypeptide as defined herein. In this context it shall benoted that the term “controlling the secretion” is intended to encompassgeneration, induction, and/or the improvement of secretion. Moreparticularly, the invention is directed to the use of a carrier domainas defined herein fused to a signal peptide as defined herein, forcontrolling the secretion of a polypeptide of interest, preferably apolypeptide as defined herein, by a host cell.

With “improvement of secretion” is meant that the amount of polypeptideof interest secreted is higher, and preferably at least 2.5, or 5 or 10%higher, than the amount obtained in the case no carrier domain fused toa signal peptide as defined herein, is used to control the secretion.

7. Non-Therapeutic Applications

In one embodiment the polypeptide of interest is an enzyme as definedherein. In such embodiment, the present application is directed tovarious non-therapeutic uses of a fusion protein according to theinvention.

In one embodiment, said polypeptide of interest preferably is an enzymeas defined herein.

In another embodiment, said polypeptide of interest preferably is aplant cell wall degrading enzyme as defined herein, and even morepreferred a cellulase as defined herein.

In more specific embodiments, the invention provides for the use of apolynucleic acid, a vector or a host cell according to the invention,for the production and secretion of a plant cell wall-degrading enzymeas defined herein, and even more preferred a cellulase of microbialorigin, as defined above, preferably of bacterial origin, and forexample from a bacteria of the genus Alteromonadaceae, e.g.Saccharophagus degradans strain 2-40, of the genus Thermomonospora,e.g., from T. fusca, of the genus Cellulomonadaceae e.g. C. fimi, of thegenus Clostridia, e.g. from Clostridium thermocellum, Clostridiumcellulolyticum, Clostridium acetobutylicum. In another embodiment thecellulase is of fungal origin, and for example from a fungus of thegenus Neocallimastigomycota, e.g. from N. patriciarum or Orpinomyces spstrain PC-2.

Even more preferred the present invention relates to the use of apolynucleic acid, a vector or a host cell according to the invention,for the production and secretion of a cellulase of C. cellulolyticum, ofC. thermocellum, of C. acetobutylicum or of Saccharophagus degradans, asdefined above in a biologically active form.

In a particularly preferred embodiment the invention relates to the useof a polynucleic acid, a vector or a host cell according to theinvention, for the production and secretion of the cellulase Cel48F ofC. cellulolyticum.

In a particularly preferred embodiment the invention relates to the useof a polynucleic acid, a vector or a host cell according to theinvention, for the production and secretion of the cellulase Cel9G of C.cellulolyticum.

In yet another particularly preferred embodiment the invention relatesto the use of a polynucleic acid, a vector or a host cell according tothe invention, for the production and secretion of the cellulase Cel5Hof Saccharophagus degradans.

8. Therapeutic Applications

A further aspect of the invention relates to the therapeutic applicationof the carrier constructs and host cells comprising carrier constructsaccording to the present invention.

In particular embodiments the polypeptide of interest is a therapeuticprotein as defined herein. In such embodiments, the present applicationis preferably directed to various therapeutic uses of a fusion proteinaccording to the invention.

It has been shown that the avascular hypoxic/necrotic regions in solidtumors which are difficult to reach with classical therapies provide asuitable environment for the growth and proliferation of obligate,anaerobic bacteria. More particularly it has been demonstrated uponintravenous injection, clostridial spores are dispersed throughout thebody, but only those that encounter the hypoxic environment of a solidtumour go on to germinate and multiply (Mose and Mose, 1964; Carey etal, 1967). Thus clostridial spores are ideal carriers for drug deliveryin cancer. However, the major problem encountered with drug-delivery byclostridia is the level of secreted proteins. The present inventionprovides a way to address this problem, by providing a system whichallows secretion of fusion proteins comprising a polypeptide of interestby micro-organisms such as Clostridia.

Accordingly, the invention provides for the use of a polynucleic acid, avector or a host cell according to the invention, for the production andsecretion of a therapeutic protein as defined herein. In particularembodiments the therapeutic protein is selected from the groupcomprising therapeutic enzymes, cytokines, and antibodies.

According to particular embodiments, the present invention relates tothe use of a polynucleic acid encoding a fusion protein comprising atherapeutic protein according to the invention and a host cellcomprising the polynucleic acid, for the production and secretion ofcytokines, more particularly cytokines in a biologically active form, byrecombinant micro-organisms.

More particularly the invention relates to the use of a polynucleic acidencoding a fusion protein comprising a therapeutic protein according tothe invention and a host cell comprising the polynucleic acid, for theproduction and secretion of a cytokine selected from the groupcomprising IL-2, IL-12, GM-CSF and TNF-α by recombinant micro-organisms.

In further embodiments the therapeutic protein is a pro-drug convertingenzyme.

In a further aspect, the invention relates to a pharmaceuticalcomposition comprising a therapeutically active amount of polynucleicacid, vector or host cell, more particularly of a recombinantmicro-organism according to the invention and at least onepharmaceutically acceptable carrier, i.e. for instance one or morepharmaceutically acceptable carrier substances and/or additives, e.g.,buffers, carriers, excipients, stabilisers, etc. More particularly, therecombinant micro-organism is in the form of a bacterial spore, mostparticularly a Clostridium spore.

The term “therapeutically effective amount” as used herein means thatamount of polynucleic acid, vector or host cell (i.a. recombinantmicro-organism or spore thererof) that elicits the biological ormedicinal response in a tissue, system, animal or human that is beingsought by a researcher, veterinarian, medical doctor or other clinician.

The term “pharmaceutically acceptable” as used herein is consistent withthe art and means compatible with the other ingredients of apharmaceutical composition and not deleterious to the recipient thereof.Suitable pharmaceutically acceptable carriers are well known to thoseskilled in the art and for instance may be selected from proteins suchas collagen or gelatine, carbohydrates such as starch, polysaccharides,sugars (dextrose, glucose and sucrose), cellulose derivatives likesodium or calcium carboxymethylcellulose, hydroxypropyl cellulose orhydroxypropylmethyl cellulose, pregeletanized starches, pectin agar,carrageenan, clays, hydrophilic gums (acacia gum, guar gum, arabic gumand xanthan gum), alginic acid, alginates, hyaluronic acid, polyglycolicand polylactic acid, dextran, pectins, synthetic polymers such aswater-soluble acrylic polymer or polyvinylpyrrolidone, proteoglycans,calcium phosphate and the like.

The pharmaceutical preparations can also contain additives, for examplefillers, disintegrants, binders, lubricants, wetting agents,stabilizers, emulsifiers, dispersants, preservatives, sweeteners,colorants, flavorings, aromatizers, thickeners, diluents, buffersubstances, solvents, solubilizers, agents for achieving a depot effect,salts for altering the osmotic pressure, coating agents or antioxidants.

The dosage or amount of an polynucleic acid, vector or host cell asdefined herein used depends on the individual case and is, as iscustomary, to be adapted to the individual circumstances to achieve anoptimum effect. Thus, it depends on the nature and the severity of thedisorder to be treated, and also on the sex, age, weight and individualresponsiveness of the human or animal to be treated, on the efficacy andduration of action of the compounds used, on whether the therapy isacute or chronic or prophylactic, or on whether other active compoundsare administered in addition to a polynucleic acid, vector or host cellas defined herein.

The preparation of the pharmaceutical compositions can be carried out ina manner known per se. To this end, the polynucleic acid, vector or hostcell as defined herein together with one or more solid or liquidpharmaceutical carrier substances and/or additives (or auxiliarysubstances) and, if desired, in combination with other pharmaceuticallyactive compounds having therapeutic or prophylactic action, are broughtinto a suitable administration form or dosage form which can then beused as a pharmaceutical in human medicine.

The pharmaceutical composition according to the invention is preferablyadministered parenterally, for example subcutaneously, intramuscularlyor intravenously in the form of solutions for injection or infusion.Other suitable administration forms are, for example, microcapsules,implants or rods.

Suitable carriers for the preparation of solutions, for example ofsolutions for injection, for example, water, physiological sodiumchloride solution, alcohols such as ethanol, glycerol, polyols, sucrose,invert sugar, glucose, mannitol, vegetable oils, etc. It is alsopossible to lyophilize the host cell as defined herein and to use theresulting lyophilisates, for example, for preparing preparations forinjection.

Suitable carriers for microcapsules, implants or rods are, for example,copolymers of glycolic acid and lactic acid.

In a particularly preferred embodiment, the pharmaceutical compositionaccording to the invention is injectable. The composition may forinstance be administered (injected) at a tumor site. Such applicationenables the delivery of the product contained in the pharmaceuticalcomposition, i.e. polynuclucic acid, vector, host cell as definedherein, to the tumor cells. The delivered compounds, e.g. polynuclucicacid, vector, host cell as defined herein, to the tumor cells, ispreferably injected one or more times a week during months, or evenyears.

In another particularly preferred embodiment, the pharmaceuticalcomposition according to the invention can be delivered usingreservoirs, such as for instance micropumps, in order to deliver theproduct, i.e. polynuclucic acid, vector, host cell as defined herein, tothe tumor in cells in cancer types.

In addition, the invention is directed to a polynucleic acid, a vector,or a host cell according to the invention for use as a medicament. Inother words, the invention also relates to the use of a polynucleicacid, a vector, or a host cell according to the invention as amedicament.

In a further embodiment, the invention relates to a polynucleic acid, avector, or a host cell according to the invention for treating cancer.More particularly, the invention relates to the use of a polynucleicacid, a vector, or a host cell according to the invention for thepreparation of a medicament for treating cancer.

In an example, recombinant clostridia bacteria engineered as disclosedherein can be used for cancer treatment, as Clostridia spores are ableto germinate and develop in the neighborhood of tumors.

Various types of cancer can be treated in accordance with the presentinvention. The invention therefore also relates to a method of treatingcancer in a subject in need thereof comprising introducing a host cellaccording to the invention in said subject, and preferably at a tumorsite in said subject. Practically, the present invention thus comprisesthe introduction of a recombinant host cell, capable of expressing apolynucleic acid according to the invention at the tumor site in asubject in need thereof; and thus capable of producing a polypeptide ofinterest according to the invention at the tumor site in the host. Thepresent invention thus provides the delivery of the product, i.e. atherapeutic protein, such as e.g. a cytokine, contained in a polynucleicacid, vector, host cell or pharmaceutical composition as defined herein,selectively into tumor cells. Based on the fact that solid tumors, atsome stage of their development, are characterized by severe hypoxia andnecrosis, such transfer system is considered as a valuable anti-cancerstrategy. Such a therapy is believed to circumvent normal tissuetoxicity and to improve tumor cell kill, as the result of its directdelivery of the product to the tumor.

The invention will be further understood with reference to the followingnon-limiting examples.

EXAMPLES

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques used in recombinant DNA technology,molecular biology, biological testing, and the like, which are withinthe skill of the art. Such techniques are explained fully in theliterature.

Example 1 Secretion of Heterologous and Toxic Cellulases

This example illustrates the secretion of polypeptides of interest, inparticular the cellulases, by C. acetobutylicum in accordance with thepresent invention. Various constructions were made and the most relevantof these are schematically represented on FIG. 1.

In a first construct, the polynucleic acid encoding the cellulase Cel48Fobtained from C. cellulolyticum, was fused to a polynucleic acidencoding a carrier domain and comprising the CBM3a module, the X module(Xc), and the cohesin 1 module of CipC of C. cellulolyticum Theconstruct further contains the signal peptide of the CipC scaffoldingprotein of C. cellulolyticum. Suitable linker sequences are used to linkthe different modules to one another.

In a second construct, the polynucleic acid encoding the cellulaseCel48F obtained from C. cellulolyticum, was fused to a polynucleic acidencoding a carrier domain and comprising the CBM3a and one X module (Xc)of CipC of C. cellulolyticum The construct further contains the signalpeptide of the CipC scaffolding protein of C. cellulolyticum. Suitablelinker sequences are used to link the different modules to one another.

In a third construct, the polynucleic acid encoding the cellulase Cel9Gobtained from C. cellulolyticum, was fused to a polynucleic acidencoding a carrier domain and comprising the CBM3a and one X module (Xc)of CipC of C. cellulolyticum. The construct further contains the signalpeptide of the CipC scaffolding protein of C. cellulolyticum. Suitablelinker sequences are used to link the different modules to one another.

As controls, a construct was made comprising a polynucleic acid encodingcellulase Cel48F obtained from C. cellulolyticum and the signal peptideof the CipC scaffolding protein of C. cellulolyticum but without acarrier domain or and X module and another construct was made comprisinga polynucleic acid encoding cellulase Cel9G obtained from C.cellulolyticum and the signal peptide of the CipC scaffolding protein ofC. cellulolyticum fused to a polynucleic acid encoding the CBM3a modulebut without the X module. In addition, similar constructs were made withand without the c-terminal dockerin domain.

The various constructs were constructed using Overlap Extension PCRtechnique, and cloned in the shuttle expression vector pSOS952, thatconfers resistance to the antibiotic erythromycin, thereby generatingthe plasmids pSOS952-CBM-Xc-Cohesin-48F; pSOS952-CBM-Xc-48F andpSOS952-CBM-Xc-9G, respectively. The constructs were checked bysequencing, and methylated in vivo. The methylated vectors weresubsequently used to electrotransform C. acetobutylicum strain ATTC 824.

It was shown that C. acetobutylicum strains bearing the first twoconstructs secreted a fusion protein containing the cellulase Cel48F intheir growth medium in amounts of about 0.5 mg/L. Furthermore, also theC. acetobutylicum strains bearing the third constructs also secreted afusion protein containing the cellulase Cel9G in its growth medium inamounts of about 0.5 mg/L. However, in the absence of the carrier domain(CBM+X module) the constructs comprising only a cellulase with a signalsequence were toxic to the cells (i.e. no secretion). The presence of ac-terminal dockerin domain did not change this.

These results show that, when the cellulases Cel9G or Cel48F are fusedby genetic engineering to the signal sequence and to two modules (CBMand X) or three modules (CBM, X and cohesin) of the scaffoldin CipC fromC. cellulolyticum, the chimeric enzymes are produced and secreted in themedium by C. acetobutylicum. The secretion yields of the engineeredcellulases were estimated to be around 0.3-0.5 mg/L. These values arebased on activity of the culture supernatant on cellulose.Alternatively, the concentration of the heterologous cellulases in theculture supernatant was also estimated by polyacrylamide gelelectrophoresis analysis under denaturing conditions followed bydensitometric analyses).

The presence of the CBM has the advantage of rapidly allowing theprotein of interest to be purified from the supernatant of the cultureon crystalline cellulose column.

Example 2 Secretion of a Heterologous and Toxic Cellulase According tothe Invention Using Multiple X Modules

This is another example illustrating the secretion of polypeptides ofinterest, in particular cellulases, by C. acetobutylicum in accordancewith the present invention. Various constructions were made and areschematically represented on FIG. 1 (lower panel), wherein modulesobtained from different scaffolding proteins were used.

In one construct the polynucleic acid encoding the cellulase Cel9Gobtained from C. cellulolyticum, was fused to a polynucleic acidencoding a carrier domain and comprising the CBM3a obtained from theCipC protein of C. cellulolyticum and one X module (Xa) obtained fromthe CipA protein of C. acetobutylicum. The construct further containsthe signal peptide of the CipC scaffolding protein of C. cellulolyticum.Suitable linker sequences are used to link the different modules to oneanother.

In a fifth construct the polynucleic acid encoding the cellulase Cel9Gobtained from C. cellulolyticum, was fused to a polynucleic acidencoding a carrier domain and comprising the CBM3a obtained from theCipC protein of C. cellulolyticum and the first (Xa) and the second(Xa′) X modules of CipA of C. acetobutylicum. The construct furthercontains the signal peptide of the CipC scaffolding protein of C.cellulolyticum. Suitable linker sequences are used to link the differentmodules to one another.

These constructs were constructed using Overlap Extension PCR technique,and cloned in the shuttle expression vector pSOS952, that confersresistance to the antibiotic erythromycin, thereby generating theplasmids pSOS952-CBM-Xa-9G and pSOS952-CBM-Xa-Xa′-9G respectively. Theconstructs were checked by sequencing, and methylated in vivo and invitro. The methylated vectors were subsequently used to electrotransformC. acetobutylicum strain ATTC 824

It was shown that C. acetobutylicum strains bearing these two constructssecreted a fusion protein containing the cellulase Cel9G in their growthmedium in relevant amounts. These results also showed that when thecellulase Cel9G is fused by genetic engineering to the signal sequenceand to the CBM of the scaffoldin CipC from C. cellulolyticum, and to oneor two X modules from CipA of C. acetobutylicum chimeric enzymes areproduced and secreted in the medium by C. acetobutylicum. This indicatesthat the X modules from CipA of Clostridium acetobutylicum also havecarrier properties with respect to secretion by C. acetobutylicum. TheApplicants also further showed that using more than one X module hadbeneficial effects on secretion. The secretion yield of the fusioncellulases was estimated at 1.9 and 3.5 mg/L for the strains carryingthe vectors pSOS952-CBM-Xa-9G and pSOS952-CBM-Xa-Xa′-9G, respectively.The concentration of the heterologous cellulases in the culturesupernatant was also estimated by polyacrylamide gel electrophoresisanalysis under denaturing conditions followed by densitometric analyses.

Example 3 Improvement of the Secretion of a Heterologous CellulaseAccording to the Invention

This is another example illustrating the secretion of polypeptides ofinterest, in particular a cellulase from Saccharophagus degradans, by C.acetobutylicum in accordance with the present invention. Variousconstructions were made and are schematically represented on FIG. 2,wherein modules obtained from different scaffolding proteins were used.

In a first construct, the synthetic polynucleic acid adapted to C.acetobutylicum codon bias and encoding the cellulase Cel5H fromSaccharophagus degradans, was fused to a polynucleic acid encoding acarrier domain and comprising the CBM3a module, the first (Xa) and thesecond (Xa′) X modules obtained from the CipA protein of C.acetobutylicum and the signal peptide of the CipC scaffolding protein ofC. cellulolyticum. Suitable linker sequences are used to link thedifferent modules to one another.

The domain structure of the native Cel5H polypeptide can be outlined asGH5-PSL-CBM6-EPR-DZ, wherein GH5 stands for its glycoside hydrolasefamily 5 domain, PSL for the polyserine linker, CBM6 forcarbohydrate-binding module family 6 domain, EPR for the glutamicacid-proline-rich region and, without being limited to thisinterpretation, DZ represents a C-terminal domain identified by thepresent inventors as a putative carbohydrate-binding module.

This construct was constructed using Overlap Extension PCR technique,and cloned in the shuttle expression vector pSOS952, that confersresistance to the antibiotic erythromycin, thereby generating theplasmid pSOS952-CBM-Xa-Xa′-5H. The constructs were checked bysequencing, and methylated in vivo and in vitro. The methylated vectorwas subsequently used to electrotransform C. acetobutylicum strain ATTC824.

The secretion by C. acetobutylicum of the wild-type Cel5H proteinappended with the signal peptide of the scaffoldin CipC from C.cellulolyticum was 0.5-0.9 mg/L (values based on the activity of theculture supernatant on para-nitrophenyl-cellobioside). However, C.acetobutylicum strains bearing the two constructs encoding the fusionprotein comprising the Cel5H protein linked to the carrier domainsecreted a fusion protein containing the cellulase 5H in their growthmedium in significantly higher amounts, more particularly up to 6.1 mg/L(value based on the activity of the culture supernatant onpara-nitrophenyl-Cellobioside, see FIG. 3). These results againdemonstrate that when the heterologous (i.e. non-Clostridial) cellulaseCel5H is fused by genetic engineering to the signal sequence and to theCBM of the scaffoldin CipC from C. cellulolyticum, and to one or two Xmodules from CipA of C. acetobutylicum chimeric enzymes are produced andsecreted in the medium by C. acetobutylicum.

Example 4 Demonstration of the Activity of the Fusion Proteins Accordingto the Invention on Cellulose

Using molecular biology techniques the DNA encoding the differentprotein constructs described in Examples 2 and 3 was amplified andcloned in an E. coli expression vector (pET22b(+), Novagen). Theresulting vector was used to transform the E. coli strain BL21 (DE3)(Novagen). In all cases, six His codons were grafted at the C-terminusextremity of the recombinant proteins to facilitate their purificationon Nickel resin (Ni-NTA, Qiagen).

The recombinant strains were grown in Luria Bertani medium and theexpression of the cloned genes was triggered using IPTG as the inducer.The synthesis of the recombinant proteins was verified by denaturingpolyacrylamide gel electrophoresis (SDS-PAGE). The cultures werecentrifuged and the harvested cells were broken in a French press.

The recombinant proteins were purified by loading the crude extract onNi-NTA (Qiagen), and elution of the protein of interest using increasingconcentrations of imidazolium. Purification was achieved using FPLCQ-sepharose (Hitrap Q HP resin, GE Healthcare).

Activity of the purified enzymes was tested on Avicel (microcrystallinecellulose) using standard conditions (37° C.). The results areillustrated in FIGS. 4 and 5 b. Alternatively, the activity was measuredon para-nitrophenyl-cellobioside and the results are presented in FIG. 5a.

Example 5 Secretion of a Therapeutic Protein

This is another example illustrating the secretion of polypeptides ofinterest, in particular a therapeutic protein interleukine 2 from rat,by C. acetobutylicum in accordance with the present invention. Aconstruction is made wherein modules obtained from different scaffoldingproteins are used.

In the construct the polynucleic acid encoding the interleukine 2 fromrat (IL2), is fused to a polynucleic acid encoding a carrier domaincomprising the CBM3a obtained from the CipC protein of C. cellulolyticumand the first (Xa) and the second (Xa′) X modules of CipA of C.acetobutylicum. The construct further contains the signal peptide of theCipC scaffolding protein of C. cellulolyticum. Suitable linker sequencesare used to link the different modules to one another.

The construct is constructed using Overlap Extension PCR technique, andcloned in the shuttle expression vector pSOS952, that confers resistanceto the antibiotic erythromycin, thereby generating the plasmidpSOS952-CBM-Xa-Xa′-IL2. The construct is checked by sequencing, andmethylated in vivo. The methylated vector iss subsequently used toelectrotransform C. acetobutylicum strain ATTC 824.

It is shown that C. acetobutylicum strains bearing this constructsecretes a fusion protein containing the rat interleukine 2 in theirgrowth medium in relevant amounts. These results also show that when therat interleukine 2 is fused by genetic engineering to the signalsequence and to the CBM of the scaffoldin CipC from C. cellulolyticum,and to two X modules from CipA of C. acetobutylicum, the chimericprotein is produced and secreted in the medium by C. acetobutylicum.This indicates that the CBM from CipC of C. cellulolyticum and the Xmodules from CipA of C. acetobutylicum also have carrier properties withrespect to secretion by C. acetobutylicum of a therapeutic protein froma mammal. The fusion protein containing the rat IL2 iss purified fromthe culture supernatant by loading the external medium on a column ofcrystalline cellulose Avicel. The fusion protein is eluted from Avicelusing purified water (milliQ water), and mass spectrometry analyses aswell as N-terminal micro-sequencing confirm the integrity of thepurified recombinant protein.

The invention claimed is:
 1. A polynucleic acid encoding a fusionprotein which comprises in this order: at least one signal peptide; acarrier domain comprising a carbohydrate binding module (CBM) of acellulosomal scaffolding protein fused to one, two, or three X modulesof a cellulosomal scaffolding protein wherein at least one X module isthe X2 module of the CipA scaffolding protein of C. acetobutylicum; atleast one peptide linker; and at least one polypeptide of interest;wherein said peptide linker links the carrier domain to the polypeptideof interest, and wherein said polypeptide of interest comprises anenzyme.
 2. The polynucleic acid of claim 1, wherein said fusion proteincomprises two or three X modules.
 3. The polynucleic acid of claim 1,wherein said signal peptide is the signal peptide of the CipCscaffolding protein of C. celluloyticum or the signal peptide of theCipA scaffolding protein of C. acetobutylicum.
 4. The polynucleic acidof claim 1, wherein said fusion protein comprises a carbohydrate bindingmodule of type-3a (CBM3a).
 5. The polynucleic acid of claim 1, whereinsaid enzyme is a cell wall degrading enzyme.
 6. The polynucleic acid ofclaim 1, wherein said enzyme is a cellulase.
 7. The polynucleic acid ofclaim 6, wherein said enzyme is a cellulase of C. celluloyticum.
 8. Thepolynucleic acid of claim 6, wherein said enzyme is C. celluloyticumCel48F or C. celluloyticum Cel9G.
 9. The polynucleic acid of claim 1,wherein said enzyme is cellulase Cel5H of S. degradans strain 2-40. 10.A recombinant micro-organism comprising the polynucleic acid encoding afusion protein according to claim 1, wherein the recombinantmicro-organism is capable of secreting said enzyme when cultured underconditions effective to cause expression of the fusion protein.
 11. Therecombinant micro-organism of claim 10, wherein said micro-organism isfrom the class of Clostridia.
 12. The recombinant micro-organism ofclaim 10, wherein said micro-organism is from a Clostridium strainselected from the group consisting of C. acetobutylicum and C.beijerinckii.
 13. The recombinant micro-organism of claim 10, whereinsaid signal peptide is the signal peptide of the CipC scaffoldingprotein of C. celluloyticum or the signal peptide of the CipAscaffolding protein of C. acetobutylicum.
 14. The recombinantmicro-organism of claim 10, wherein said fusion protein comprises acarbohydrate binding module of type-3a (CBM3a).
 15. The recombinantmicro-organism of claim 10, wherein said enzyme is a cell wall degradingenzyme.
 16. The recombinant micro-organism of claim 10, wherein saidenzyme is a cellulose of C. cellulolyticum.
 17. The recombinantmicro-organism of claim 10, wherein said enzyme is cellulase Cel5H of S.degradans strain 2-40.
 18. A method for the production and secretion ofan enzyme of interest comprising the steps of: introducing thepolynucleic acid encoding the fusion protein according to claim 1 into arecombinant micro-organism, wherein the polypeptide of interestcomprises the enzyme of interest; and culturing said recombinantmicro-organism under conditions effective to cause expression of thefusion protein, wherein the enzyme of interest is secreted by therecombinant micro-organism into the culture medium of said recombinantmicro-organism.
 19. The polynucleotide of claim 1, wherein thecarbohydrate binding module is fused to one X module of a cellulosomalscaffolding protein, wherein said X module is the X2 module of the CipAscaffolding protein of C. acetobutylicum.
 20. The recombinantmicro-organism of claim 10, wherein the fusion protein comprises acarbohydrate binding module fused to one X module of a cellulosomalscaffolding protein, wherein said X module is the X2 module of the CipAscaffolding protein of C. acetobutylicum.